Rab GTPases are highly conserved components of vesicle trafficking pathways. Rab5, as a master regulator of endocytic trafficking, has been shown to function in membrane tethering and docking. However, the function of Rab5 in meiosis has not been addressed. Here, we report elongated spindles and misaligned chromosomes, with kinetochore-microtubule misattachments, on specific depletion of Rab5a in mouse oocytes. Moreover, the localization and levels of centromere protein F (CENPF), a component of the nuclear matrix, are severely reduced at kinetochores in metaphase oocytes following Rab5a knockdown. Consistent with this finding, nuclear lamina disassembly in the transition from prophase arrest to meiosis I is also impaired in Rab5a-depleted oocytes. Notably, oocytespecific ablation of CENPF phenocopies the meiotic defects resulting from Rab5a knockdown. In summary, our data support a model where Rab5a-positive vesicles, likely through interaction with nuclear lamina, modulate CENPF localization and levels at centromeres, consequently ensuring proper spindle length and kinetochore-microtubule attachment in meiotic oocytes.